Abstract
BackgroundCyclooxygenase-2 (COX-2), the inducible COX form, is a bi-functional membrane-bound enzyme that typically metabolizes arachidonic acid (downstream ω-6 fatty acid) to form 2-series of prostaglandins known to be involved in cancer development. Overexpression of COX-2 has been found in a majority of breast carcinomas, and has also been associated with increased severity and the development of the metastasis. Our lab recently demonstrated that COX-2 can also metabolize dihomo-γ-linolenic acid (DGLA, a precursor of ω-6 arachidonic acid) to produce an anti-cancer byproduct, 8-hydroxyoctanoic acid (8-HOA) that can inhibit growth and migration of colon and pancreatic cancer cells. We thus tested whether our strategy of knocking down delta-5-desaturase (D5D, the key enzyme that converts DGLA to arachidonic acid) in breast cancer cells overexpressing COX-2 can also be used to promote 8-HOA formation, thereby suppressing cancer growth, migration, and invasion.MethodsSiRNA and shRNA transfection were used to knock down D5D expression in MDA-MB 231 and 4 T1 cells (human and mouse breast cancer cell lines expressing high COX-2, respectively). Colony formation assay, FITC Annexin V/PI double staining, wound healing and transwell assay were used to assess the effect of our strategy on inhibition of cancer growth, migration, and invasion. GC/MS was used to measure endogenous 8-HOA, and western blotting was performed to evaluate the altered key protein expressions upon the treatments.ResultsWe demonstrated that D5D knockdown licenses DGLA to inhibit growth of breast cancer cells via promoting formation of 8-HOA that can inhibit histone deacetylase and activate cell apoptotic proteins, such as procaspase 9 and PARP. Our strategy can also significantly inhibit cancer migration and invasion, associated with altered expression of MMP-2/− 9, E-cadherin, vimentin and snail. In addition, D5D knockdown and DGLA supplementation greatly enhanced the efficacy of 5-fluorouracil on breast cancer growth and migration.ConclusionsConsistent to our previous studies on colon and pancreatic cancer, here we demonstrate again that the high level of COX-2 in breast cancer cells can be capitalized on inhibiting cancer growth and migration. The outcome of this translational research could guide us to develop new anti-cancer strategy and/or to improve current chemotherapy for breast cancer treatment.
Highlights
Cyclooxygenase-2 (COX-2), the inducible COX form, is a bi-functional membrane-bound enzyme that typically metabolizes arachidonic acid to form 2-series of prostaglandins known to be involved in cancer development
Consistent with the results demonstrated in colon and pancreatic cancer cells [46,47,48,49], both direct 8-hydroxyoctanoic acid (8-HOA) treatment and endogenous 8-HOA resulting from D5DKD and Dihomo-γ-linolenic acid (DGLA) treatment in MDA-MB 231 cells upregulated acetyl-histone H3 (AcH3) and γH2AX (DNA damage marker, Fig. 8a), indicating that 8-HOA can suppress cancer cell growth by inhibiting Histone deacetylase (HDAC) and inducing DNA damage
Our study demonstrated that D5D-KD along with DGLA treatment can promote formation of 8-HOA which serves as an histone deacetylase inhibitor (HDACi) to induce DNA damage, and inhibit cancer cell growth and migration, via regulating key signaling proteins, e.g., MMPs-2, 9, e-cadherin, etc, Fig. 10)
Summary
Cyclooxygenase-2 (COX-2), the inducible COX form, is a bi-functional membrane-bound enzyme that typically metabolizes arachidonic acid (downstream ω-6 fatty acid) to form 2-series of prostaglandins known to be involved in cancer development. We tested whether our strategy of knocking down delta-5-desaturase (D5D, the key enzyme that converts DGLA to arachidonic acid) in breast cancer cells overexpressing COX-2 can be used to promote 8-HOA formation, thereby suppressing cancer growth, migration, and invasion. COX-2 substrates, including eicosapentaenoic acid and docosahexaenoic acid (two ω-3 fatty acids) and dihomo-γ-linolenic acid (perhaps an exceptional ω-6 fatty acid), have shown some inhibitory effects on cancer cell growth and metastasis, most likely due to their competition with AA for the COX-2 peroxidation to limit PGE2 formation [6,7,8,9,10,11,12, 20,21,22,23]. COX-2 inhibitors are ineffective in general, and suffer from critical safety issues in patients such as increased risks of cardiovascular disease and gastrointestinal tract injury [39,40,41,42]
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